Ulrike Bacher

Conventional cytogenetics of myeloproliferative diseases other than CML contribute valid information.

In chronic myeloproliferative disorders other than CML (CMPD) recurrent cytogenetic abnormalities occur, but specific patterns of chromosomal aberrations in the specific entities have so far not been detected. Thus, the value of conventional cytogenetics in the routine diagnostic setting of CMPD remains to be clarified. We performed a cytogenetic study on 409 patients with different CMPD [polycythemia vera, essential thrombocytosis (ET), idiopathic osteomyelofibrosis, chronic myelomonocytic leukemia (proliferative subtype), idiopathic hypereosinophilic syndrome (HES), myeloproliferative syndrome (unclassifiable)] and on 102 patients with suspected CMPD. Cytogenetic abnormalities occurred in different frequencies ranging from 3 to 40% depending on the subtype, and showed some specific differences with respect to their type. The highest frequency and the most complex pattern of clonal aberrations were observed in idiopathic osteomyelofibrosis. However, clonal aberrations were also found in 10% of patients with suspected CMPD establishing the diagnosis of a malignant disease. In conclusion, cytogenetics are essential in the routine diagnostic setting of CMPD or cases suspicious for CMPD. In ET and in HES the aberration rate was only 3 and 7%, respectively. Thus, cytogenetics can be omitted. However, in some of these cases molecular procedures should be integrated into the routine diagnostic process.

Further correlations of morphology according to FAB and WHO classification to cytogenetics in de novo acute myeloid leukemia: a study on 2,235 patients.

In routine diagnostic procedures of acute myeloid leukemia (AML), the French–American–British (FAB) and World Health Organization (WHO) classifications both play a central role. Some morphologic subtypes are specifically associated to distinct cytogenetic and molecular aberrations; however, such close correlations do not exist for the majority of entities. We evaluated cytogenetics in 2,235 patients at diagnosis of AML with the FAB subtypes M0–2, M4, and M5–7. The cytogenetic patterns of these subtypes showed differences with respect to the clonal aberration rate and the incidence of complex aberrant karyotypes. The frequency of numerical gains and losses and of structural losses and the incidence of 11q23/MLL rearrangements differed. Thus, cytomorphology of AML may be helpful to support or even initiate other diagnostic procedures, e.g., interphase fluorescence in situ hybridization and polymerase chain reaction. In conclusion, the central role of morphology as defined by the FAB and WHO classification in AML at diagnosis is still justified in combination with other techniques.

Evaluation of complete disease remission in acute myeloid leukemia: a prospective study based on cytomorphology, interphase fluorescence in situ hybridization, and immunophenotyping during follow-up in patients with acute myeloid leukemia.

Different diagnostic methods add information to define complete remission (CR) in patients with acute myeloid leukemia (AML). The detection of minimal residual disease (MRD) for predicting prognosis and for therapeutic planning still are under discussion.

Discrimination of chronic lymphocytic leukemia (CLL) and CLL/PL by cytomorphology can clearly be correlated to specific genetic markers as investigated by interphase fluorescence in situ hybridization (FISH)

Although interphase fluorescence in situ hybridization (FISH) is routinely used in chronic lymphocytic leukemia (CLL), differences in the chromosomal pattern with respect to morphological subtypes of CLL (typical CLL, CLL/PL, PLL) are still under debate. We studied 153 patients with CLL and correlated cytomorphology on peripheral blood stains with FISH analysis and other prognostic markers. The percentage of prolymphocytes was calculated as a continuous variable and followed published thresholds in parallel while being correlated to FISH analysis. Higher percentages of prolymphocytes were associated significantly with deletion of 17p13. Deletion of 17p13 was most frequently observed in patients with more than 30% prolymphocytes. Trisomy 12 was found mainly in cases with 6–30% prolymphocytes. The percentage of prolymphocytes did not correlate with deletions of 11q23 or with 13q14 abnormalities. In conclusion, we suggest that further research focus on the percentage of prolymphocytes in CLL. Doing so, biologically relevant thresholds for the percentages of prolymphocytes in the peripheral blood and their association to underlying genetic markers could be investigated together with other biologically and especially prognostic markers.

Blast count and cytogenetics correlate and are useful parameters for the evaluation of different phases in chronic myeloid leukemia

Staging of chronic myeloid leukemia (CML) phases is based on cytomorphological criteria that vary considerably between different staging systems. Thus, staging of CML is heterogeneous and causes problems with respect to the comparison of therapeutical strategies and clinical outcome. We evaluated 59 patients with CML in different stages of the disease. In order to define which cytomorphological parameters correlate with cytogenetics we investigated cytomorphology and cytogenetics in parallel in all cases. As a result, bone marrow blast count demonstrated a highly significant correlation with the respective cytogenetic results of the patients and was clearly linked to the frequency and complexity of clonal evolution. We therefore propose to focus staging systems of CML on the correlation of the percentage of bone marrow blasts and the cytogenetic results.

The complex isotopologue space of glucose as a framework for the study of human intermediary metabolism.

The positional distributions of stable isotopes in metabolites provide specific fingerprints of the pathways and fluxes that have occurred in the organisms under study. In particular, modern nuclear magnetic resonance (NMR) spectroscopy enables the detailed assignment of isotope patterns in natural products, for example, in metabolites obtained from labelling experiments using 13C-enriched precursors, such as glucose, acetate or CO2. In this study, the transient 13C-isotopologue composition of blood glucose from an adult human volunteer after intravenous supply of [U-13C6]glucose was determined by high-resolution 13C NMR spectroscopy. The non-linear progression curves displaying the relative amounts of eight 13C-glucose isotopologues reflected the contributions of glucose metabolism by glycolytic cycling, the pentose phosphate pathway and anaplerotic reactions involving the citric acid cycle. The pilot study suggests that the experimental setting can be useful in analysing under non-invasive conditions the impact of physiological and pharmacological constraints on glucose turnover in humans.

Erythroblastic synartesis in a patient initially diagnosed with myelodysplastic syndrome.

Malfunction of erythropoiesis in elderly patients is often caused by myelodysplastic syndrome (MDS). However, in patients with low red blood counts, other causes of bone marrow dysfunction have to be considered. An uncommon disorder is erythroblastic synartesis. Since its first description in 1973 only one further series of three patients has been published [1, 2]. Erythroblastic synartesis is considered to be caused by autoimmunological mechanisms, leading to aggregation of erythroid cells and subsequently to anemia. We report a case of erythroblastic synartesis initially diagnosed as MDS. In March 2000, a 74-year-old male patient presented with weakness and loss of physical strength. Clinical history and physical examination were uneventful. Routine blood count revealed a hemoglobin level of 9.0 g/dl, whereas leukocyte and thrombocyte counts were normal. Bone marrow biopsy showed hyperplastic erythropoiesis with some dysplastic features and less than 5% myeloblasts. Refractory anemia as a subtype of MDS according to the French-American-British Cooperative Group (FAB) classification was suspected and supportive treatment with blood transfusions every 4 weeks was initiated. In July 2003, the patient was admitted to our institution for a second opinion. Lack of physical strength was still the major complaint. Laboratory analysis showed normochromic and normocytic anemia with a hemoglobin level of 8.4 g/dl and a reticulocyte count of 15‰. There were no signs of hemolysis. Leukocyte and thrombocyte counts were normal as well as differential blood count and red cell morphology. Bone marrow biopsy was performed and cytomorphological analysis revealed increased cellularity and hyperplastic erythropoiesis. Apart from binuclear erythroid cells no dysplastic features were found. However, clusters of closely linked erythroid cells of different maturation stages were detectable (Fig. 1). Megakaryocytes and granulopoietic cells as well as lymphocytes were normal in numbers and morphology. The absence of dysplastic features and the presence of clusters of erythroid cells did not support the diagnosis of MDS. Consequently, we considered other causes. Increased erythropoiesis with clusters of erythroid cells in combination with nonbasophilic areas at sites of close proximity between erythroblasts is specific for an uncommon disorder: erythroblastic synartesis. Erythroblastic synartesis is characterized by dyserythropoiesis with clusters of erythroid cells linked by interdigitating processes and is reported to be associated with lymphoproliferative or autoimmunological diseases. In the largest series of patients with erythroblastic synartesis, Cramer et al. described three cases. Two of them were diagnosed with indolent B-cell lymphoma (one small lymphocytic lymphoma and one chronic lymphocytic leukemia). In addition, one patient suffered from

TP53 Mutations in Acute Myeloid Leukemia

Mutations of the TP53 gene show a low frequency in overall acute myeloid leukemia (AML). However, they were found at frequencies of 60–80 % in complex karyotype AML, and are strongly associated with therapy-related AML. TP53 mutations are considered to represent a separate functional category independent from the typical class I and class II mutations. The mutations are heterogeneous and are distributed across the TP53 gene with clustering of the mutations in exons 5–8. TP53 mutations confer an adverse prognostic impact in patients with AML. High-throughput sequencing facilities are now available for rapid screening for TP53 mutations at diagnosis of AML aiming to identify patients who may have a benefit from early allogeneic hematopoietic stem cell transplantation or other alternative therapeutic approaches.

Microarray-Genexpressionsanalysen in der Leukämiediagnostik

ZusammenfassungBislang basierte die Diagnostik bei Leukämien auf der Kombination verschiedener Methoden, wobei insbesondere den zytogenetischen und molekularen Techniken neben der Morphologie und der Immunphänotypisierung ein besonderer Stellenwert zukam. Mit diesem aufwendigen multimodalen Ansatz wurde es möglich, einen Großteil der Leukämiefälle in biologisch und prognostisch definierte Subgruppen einzuteilen und für einige Subtypen gezielte Therapiekonzepte zu entwickeln. Heute ermöglichen Genexpressionsanalysen mit Microarrays die simultane Analyse des Expressionsstatus zehntausender Gene in kürzester Zeit und sind daher hervorragend geeignet, die Klassifikation der Leukämien im Hinblick auf Genauigkeit, Sicherheit, Schnelligkeit und Effizienz zu optimieren. Ferner wird dieser Ansatz zur Definition neuer klinisch und prognostisch relevanter Subgruppen auf der Basis von Genexpressionssignaturen führen, damit eine wesentliche Grundlage für individuellere Therapieentscheidungen darstellen und das Verständnis für die Pathogenese der Leukämien verbessern. Eine Verwendung in der Routinediagnostik wird derzeit in einer großen internationalen Studie geprüft. Dazu ist parallel ein intensiver Vergleich mit den Standarduntersuchungsverfahren notwendig. Hier sollen der Status quo und die Perspektiven dargestellt werden.AbstractSo far, the diagnosis in leukemia is based on cytomorphology and cytochemistry, multiparameter flow cytometry, combined with cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction (PCR). This allows classification and definition of biologically defined and prognostically relevant subtypes in nearly all cases. It led also in same subgroups to targeted treatment approaches. Today, microarray technology enables us to quantify simultaneously the expression status of many ten thousands of genes in one single experiment. This novel tool seems able to renew the diagnosis and classification in leukemia and may even become essential for the molecular classification of leukemias in the near future. An ongoing international study (MILE study) is testing the accuracy, sensibility and specificity of gene expression profiling in leukemia diagnostics. First results and future directions will be given in the article.